PI3KC2a is a broadly expressed lipid kinase with critical functions during embryonic development but poorly defined roles in adult physiology. Here we utilize multiple mouse genetic models to uncover a role for PI3KC2a in regulating the internal membrane reserve structure of megakaryocytes (demarcation membrane system) and platelets (open canalicular system) that results in dysregulated platelet adhesion under haemodynamic shear stress. Structural alterations in the platelet internal membrane lead to enhanced membrane tether formation that is associated with accelerated, yet highly unstable, thrombus formation in vitro and in vivo. Notably, agonist-induced 3-phosphorylated phosphoinositide production and cellular activation are normal in PI3KC2a -deficient platelets. These findings demonstrate an important role for PI3KC2a in regulating shear-dependent platelet adhesion via regulation of membrane structure, rather than acute signalling. These studies provide a link between the open canalicular system and platelet adhesive function that has relevance to the primary haemostatic and prothrombotic function of platelets. ? 2015 Macmillan Publishers Limited. All rights reserved.